The use of optical methods for imaging and treatment of cancer is growing rapidly, largely because they work well in the setting of early detection methods, treatment of pre-malignancies or as an adjuvant treatment. Optically active agents can provide a molecular-specific or site-specific localization in tumors, and in this work we exploit both of these for diagnostic and therapeutic use. In particular, we focus on aminolevulinic acid, which induces production of protoporphyrin IX (PPIX) in the mitochondria of cells, thereby providing both a diagnostic measure of cellular metabolism as well as a way to strategically cause photodynamic damage at this site. We will use two technologically advanced approaches to quantifying the fluorescence in vivo, including our tissue micro-sampling probe and our near-infrared tomographic instrumentation. We will systematically examine the optimal way to measure fluorescence from PPIX in tumors, using needle measurements, diffuse probe measurements and diffuse tomography imaging of tissue. We will use the optimal solution in several specific biological hypotheses. First in diagnostic use, we will examine how the production of PPIX is related to the growth rate of cancer cells, focusing on the multiple cell lines available in the Dunning prostate tumor. This will also help to explore the feasibility of using repeated dosing in imaging, and examining how this affects the measurements. For therapeutic targeting, we will use the imaging system to help us explore a novel targeting approach that we have developed, which synergistically combines photodynamic pre-treatment with radiation therapy. We have found that damage to the mitochondria causes acute respiration shut down, reducing the consumption of oxygen and providing a good way to increase the oxygenation in tumors. This effect was combined with single dose radiation therapy and was found to increase the efficacy of tumor destruction. In this study, we will further examine this effect focusing on exploiting the potential for fractionated delivery of both therapies. Since ALA-PPIX provides a proportionate measure of the cellular metabolic rate, we will use the fluorescence from PPIX as a diagnostic measure of cellular activity, while using indocyanine green fluorescence as a measure of the vascular volume of the tissue. Both can be quantified in the same tissue by appropriate filtering and sequencing of the light signals, and this combined imaging approach will be examined for reliability of monitoring vascular versus cellular damage in vivo.